Movement, behavior and higher brain function all depend upon the ability of neurons to communicate via specialized intercellular junctions called synapses. Many forms of neurological disease affect the synapse both in the central nervous system (CNS: e.g. Parkinson's Disease, Epilepsy) and at the neuromuscular junction (NMJ: e.g. Myasthenia and several types of Muscular Dystrophy). Moreover, synapse regeneration is central to neural repair following brain trauma (e.g. stroke) or injury. (e.g. loss of a limb). Our objective is the systematic dissection of the function and organization of individual components of the synapse. Such an analysis requires methods to identify specific synaptic proteins as well as methods to assay their function. Drosophila offers unique possibilities for a rigorous analysis of this kind: sophisticated genetic methods can be combined with refined functional and anatomical assays to study an assessable synapse, the NMJ. This proposal has two specific aims: 1) to identify new proteins involved in presynaptic function and development by performing genetic screens and, 2) to characterize the phenotype of existing mutants in order to define their role in presynaptic mechanisms. For the first objective, screens will be conducted by identifying mutants with defective NMJs among uncoordinated lethal mutations on the third chromosome (40% of the genome). Our aim is to saturate the third chromosome for synapse dysfunction mutants, order these mutants into functional classes and initiate a molecular characterization of the isolated genes. For the second objective, we will focus on the phenotypic characterization of known synaptic mutants isolated via reverse genetic techniques. This work will include the functional and anatomical characterization of mutants in Neurexin, a Rab3A-interacting gene and double-mutant combinations of presynaptic genes. Both forward and reverse genetic approaches demand assays that measure different aspects of defective synaptic function in clear and quantifiable ways. We have developed assays to monitor the developing embryonic NMJ either in vivo or in culture using a combination of patch-clamp electrophysiology, gross morphology and ultrastructural analyses. The intention of this proposal is to bring these approaches together in order to increase substantially the number of genes known to be required at the synapse. In the long term, we intend to mutate the entire genome to identify and describe the genetic and molecular pathways directing the construction and working of the synapse.